The functional organization of small eukaryotic chromosomes has traditionally been investigated with complementary molecular and classical genetic approaches. However, the relatively large size of mammalian genomes requires that a small subchromosomal region be studied in order to narrow the gap between the two approaches. The 5 centimorgan region of DNA surrounding the mouse agouti locus is a model system for investigating 1) the relationship between genetic and physical distance; 2) the molecular basis of cis-mediated position effects on gene expression; and 3) the arrangement of transcriptional units along the chromosome with regard to inter-unit spacing, chromatin condensation, and timing of DNA replication. This region of DNA contains a number of genes that affect early embryogenesis, as well as two endogenous proviral insertion sites (Emv-13 and Emv-15), the parotid secretory protein gene (Psp) and the proto- oncogene c-src. Long range restriction maps of the Psp-agouti interval (1200 kb) and the Emv-15-c-src interval (2200 kb) will be linked together along with the Emv-13 probe to encompass an estimated 10,000 kb region. A series of additional probes within the region will be isolated by a novel strategy based on enzymatic amplification of circularization or "jumping" fragments. Because the sequence of only one end of each fragment will be known, the other end will be recovered by ligation of a marker oligonucleotide and subsequent amplification with a complementary primer. A high resolution physical map of the region, including the position of chromosomal rearrangements that act via a cis-mediated position effect, will be determined by pulsed field gel electrophoresis, and the relationship of physical to genetic distance will be studied with interspecific crosses. A sensitive in situ mapping technique will be used to study chromatin condensation for selected sets of probes by comparing distance in interphase nuclei to distance in kilobases and will be compared to the timing of DNA replication. These efforts will establish how the spacing of transcriptional units, DNA replicons, and chromatin domains are related to one another over an extended mammalian subchromosomal segment and will ultimately provide molecular access to a number of genes that affect developmental processes in mouse and man.